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Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a gut-derived metabolite, with adverse outcomes of CVD. As of today, TMAO's effects on cardiac structure and function are not well understood. In this study, both male and female TMAO-treated hearts showed functional deficits based on electrocardiography and echocardiography results. Immunohistochemistry results showed signs of hypertrophic cardiomyopathy in TMAO-treated male hearts while female TMAO-treated hearts showed signs of dilated cardiomyopathy. Neither TMAO group showed signs of fibrosis. Overproduction of reactive oxygen species was only observed in male TMAO-treated hearts. At the level of individual cardiomyocytes, significant delays in time to reach maximum contraction and dilation were only seen in TMAO-treated male hearts along with higher contractile force. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to comprehensively characterize features of TMAO-induced heart failure in both males and females which extends our current knowledge from human clinical associations. / Master of Science / Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a compound derived from eggs, red meat and seafood, with adverse outcomes of CVD. As of today, TMAO's impact on the heart is not well understood. After supplementing mice with TMAO, we discovered deficiencies in heart function coupled with altered heart structure showing signs of hypertrophic cardiomyopathy in males and dilated cardiomyopathy in females. In-depth experiments suggest that TMAO-induced cell stress could be a potential underlying cause of previously mentioned changes but the specific mechanisms require further investigation. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to characterize features of TMAO-induced heart failure aiming to unravel relevant biological changes in both male and female mice which extends our knowledge from human clinical associations.

The main objectives of this study were to investigate the applicability of chitosan as an effective wood preservative against subterranean termites, conduct metagenomic analysis of the bacterial hindgut community of Reticulitermes flavipes exposed to chitosan-treated wood, and perform chitosanase activity assay of metagenomics suggested bacterial species potentially responsible for chitosan breakdown. Chitosan showed termiticidal effects on subterranean termites at varying retention levels. Termite mortality increased when exposed to samples treated with higher chitosan concentration solutions. Approximately 40 - 100% of chitosan retained in treated-wood was leached depending on the initial retention. Post-leaching results indicate chitosan is not suitable for protection against both subterranean termites in outdoor conditions, but should be effective in non-leaching/indoor applications. For metagenomic analysis of the bacterial hindgut community of Reticulitermes flavipes, two methods were used for sequence data interpretation. The Illumina BaseSpace program identified twenty-six bacteria phyla with significant differences in abundance between the chitosan-treated and control groups. The second method, mothur, identified fifteen bacterial phyla also with significant differences in abundance between both treatment groups. Similar bacterial taxa were uniquely assigned to samples from termites fed on chitosan-treated wood using both methods. These results suggest a treatment driven effect on the hindgut bacteria diversity. While majority of the bacterial taxa were common to both methods, inconsistencies detected using the BaseSpace program suggests that the Greengenes database in its present state is not reliable for 16S rRNA gene sequence analysis. As for chitosanase activity of bacterial species with significance abundance from chitosan-treated wood exposed termites, three bacteria species, Lactococcus raffinolactis, Lactococcus lactis, and Dysgonomonas gadei, were examined. After culturing on chitosan media plates and broth, no conclusive activity could be detected from all three species. Further studies need to be conducted to understand the mechanism of chitosan toxicity to termites and insects in general and to prevent chitosan leaching from treated wood. A comparative metatransciptomic study needs to be implemented to supplement the metagenomic study performed herein, so as to elucidate the exact bacteria species involved in chitosan breakdown and the enzymes produced. Also, other bacterial species suggested by the metagenomic data to possess chitosanase activity should be investigated.

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京都大学 / 新制・課程博士 / 博士(理学) / 甲第23749号 / 理博第4839号 / 新制||理||1692(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 半谷 吾郎, 教授 湯本 貴和, 教授 古市 剛史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The collection of microbes that inhabits the human gastrointestinal tract is known as intestinal microbiota, and an enormous body of work has shown that their activities contribute to health and disease. Ulcerative colitis (UC), which is a type of inflammatory bowel disease, is considered to arise due to a disruption in the balance between the immune system and microbiota. However, there is little consensus on the mechanism of action and microbes involved in the disease manifestation. In this work, I applied culture-enriched metagenomics (CEMG) to characterize the dynamics of gut microbiota in healthy individuals and UC patients. I showed that CEMG provides a higher resolution to study these microbial communities, and we used this approach to understand microbial colonization after fecal microbiota transplantation (FMT) therapy in UC patient. I showed that sequencing approaches alone did not reveal consistent engraftment across FMT responders. Using CEMG and a collection of bacterial whole-genome sequences, I showed patient-specific microbial strain transfer and a signature of commonly engrafted genes only in patients who responded to FMT. In this work, I also investigated the dynamics of a highly abundant bacteriophage, crAssphage, in an FMT donor and implemented a new method to detect bacteriophage engraftment post-FMT using SNP analysis. Finally, it has been suggested that antibiotic treatment before FMT may increase the efficacy of FMT. However, in this work, I show that while antibiotics alter the microbiome, there was no difference in the composition of the microbiome of antibiotic vs placebo group post-FMT. This is consistent with the randomized controlled trial results that shows pretreatment with antibiotics does not improve FMT outcome. Together, this work demonstrate the importance of in-depth microbiome analysis applied to culture-dependent and -independent sequencing to characterize microbial changes post-FMT. / Dissertation / Doctor of Philosophy (PhD) / Many bacteria reside in the human gut, and they are essential in our health and in disease. It is evident that these bacteria are associated with inflammatory bowel disease, but we do not yet know how and what bacteria are involved in this disease. In this work, I describe a method to study these bacteria from stool that relies on growing them and investigating their DNA. I showed that our approach helped us recover a greater diversity of these bacteria and their genetic content in healthy individuals and patients with inflammatory bowel disease compared to methods that use only DNA based approaches. Using this method, we could better understand why some patients responded to a treatment consisting of transferring stool content from healthy donor to patient. I also investigated a group of viruses that infect bacteria and implemented a new computational method based on DNA sequencing to test whether these viruses transfer to the patient after receiving the fecal therapy. We also found that antibiotic treatment before fecal therapy in patients with inflammatory bowel disease does not improve the patient’s recovery.

Background and Research Aim: Stress affects the immune system, which influences host physiology. Mast cells have been associated with several stress-induced changes in gut physiology. Mast cells also have the potential to influence the brain and behaviour. We investigated how mast cells influenced the body, brain, and behaviour during stress. Methodology: We investigated the behaviour of mast cell deficient animals and deficient animals that received whole bone marrow (WBM) transplants. We also studied the effects of mast cell stabilization during stress on changes in gut motility, via ex vivo. recordings of intestinal segments, and brain, via behavioural measurements and flow cytometry analysis of proinflammatory monocyte trafficking to the brain. Results: Mast cell deficiency leads to several behavioural changes related to activity level, exploration, and sociability. Furthermore, deficient animals that received WBM transplants demonstrated social and anxiety-like behaviour that differed from their deficient counterparts. Mast cell stabilization during stress prevented many of the stress-induced changes in gut motility commonly observed in the intestine. Mast cell stabilization during stress also prevented proinflammatory monocyte trafficking to the brain and was associated with reduced anxiety-like behaviour. Conclusion: Our findings support the role of mast cells in baseline behaviour, suggesting the presence of mast cells is needed for normal social and anxiety-related functioning. We also found that mast cell activation contributes to stress-induced intestinal dysmotility, suggesting that mast cells should be a target for interventions of stress-related gut motility disorders, such as irritable bowel syndrome. Lastly, our findings on the role of mast cells in monocyte trafficking and anxiety adds to our knowledge of neuroimmune interactions during stress and supports a potential role for mast cells in anxiety-related mood disorders, where stabilization of mast cells during stressful events may be of benefit. / Thesis / Master of Science (MSc) / Stress affects the immune system, which influences the body. Mast cells of the immune system are involved in several stress-induced changes in the body. They can also influence the brain and behaviour. We investigated how mast cells influence the changes that occur in the gut, brain, and behaviour during stress. Using mouse models, we prevented mast cells from activating during stress and looked at this effect on gut movement, changes in brain chemistry, and behaviour. We also compared the behaviour of mast cell deficient mice and deficient mice that gained mast cells. We found that by preventing mast cells from activating during stress, we can prevent several stress-associated changes in gut movement, brain chemistry, and anxiety behaviour. We also found that mast cells affect anxiety and social behaviour. These results suggest that mast cells impact the body, brain chemistry, and behaviour in stress and non-stress conditions.

Background: The introduction of solid foods is an important dietary event during infancy and is associated with a time of dramatic shifts in gut microbial composition. The influence of solid food introduction on gut bacterial dynamics remains understudied. Methods: 15 healthy, full-term, vaginally born, and breast-fed infants of the Baby, Food and Mi sub-study of the Baby & Mi Study were investigated. Caregivers were asked to collect daily stool samples and food diaries for 17 days, commencing three days prior to the introduction of solids. Additional stool samples were available up to one year as part of the Baby and Mi study. The exposure of interest, nutritional patterns, was analyzed using food composition output from ESHA’s Food Processor. The number of food items and food groups introduced were used to calculate dietary diversity scores. The outcome of interest, gut bacterial dynamics, was analyzed using RStudio. Results: The mean (SD) age at the introduction of solid foods is 5.5 (0.66) months (n = 15). Over the study period, the proportion of estimated energy intake from solid foods was low (7.5%; SD 6.74%) (n = 14). Alpha diversity increased over time and was highest at 1 year. The gut microbial community influenced by dominant bacterial taxa changed with increasing age. With introduction of solids, individual community composition changed, though to a varying extent. Shannon alpha diversity was directly associated with calories from carbohydrates, particularly daily fiber intake. The infant’s dietary diversity score was directly associated with alpha diversity and was also positively associated with the degree of change occurring in this time period. Conclusion: Fiber intake and the dietary diversity scores had the closest relationships to the gut microbiome’s alpha diversity and community structure in infants at the time of solid food introduction. / Thesis / Master of Science (MSc) / The introduction of solid foods is an important life-event during infancy. This is also when the gut microbiome is developing to its mature state. Since nutrition is an important factor influencing the microbiome, investigating the dietary choices at the introduction to solid foods is the aim of the following study. Here, daily stool samples and food diary entries were collected for 15 healthy, breast-fed infants. It is important to measure the diversity of the bacteria in the gut of an individual (alpha) and between people (beta), as well as bacteria present. Carbohydrates drive the change in alpha diversity, especially fiber. Feeding infants a diet with many different foods shows increased alpha diversity and change in the microbiome immediately after introduction. Interestingly, the infant gut microbiome reacts to fiber in a manner comparable to the adult gut microbiome, i.e. increased bacterial diversity, which is associated with better health outcomes in adults.

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